The PI proposes an innovative approach to generate enriched populations of dopaminergic (DA) neurons for cell therapies. Idiopathic death of DA neurons causes the symptoms of Parkinson's disease (PD), thus, replacement of these cells is a primary goal of PD research. Development of strategies to generate robust cultures of DA neurons within suitable environments to support viability after implantation is imperative. In addition, implanted cells are generally a mixed population of NPCs at various stages of development, which has been reported to negatively impact therapeutic efficacy and result in side effects. Here, a method is proposed for separation of DA precursors from ventral mesencephalic neural progenitor cells (VM NPCs) prior to ex vivo expansion and differentiation in 3D, hyaluronic acid (HA) hydrogels, which serve as biomimetic culture environments and possibly as vehicles for cell transplantation. Specific Aim 1 will exploit the specific interactions between the cell surface receptor CD44 and the glycosaminoglycan HA. Both HA and CD44 are over expressed in fetal brain and down regulated during development. Preliminary results in the PI's lab demonstrate that HA-coated surfaces seeded with mixed cultures selectivity bind immature NPCs. HA-coated surfaces of varying concentrations will be used to pan for DA precursors from primary isolations of VM NPCs. Qualitative analysis of protein expression via immunostaining and quantitative analysis of mRNA expression via RT-PCR will be used to characterize NPCs at different stages of differentiation. In particular, enriched population of DA precursors will be identified. CD44 expression of NPC populations will be analyzed using fluorescence-activated cell sorting (FACS) and threshold values of CD44 expression for different NPC populations will be established. In Specific Aim 2, pre-separated NPCs enriched in DA precursors will be cultured in 3D, HA hydrogels previously developed by the PI to enhance differentiation of VM NPCs into neurons. These hydrogels have been designed to provide a biomimetic environment in which the mechanical and chemical properties closely resemble those of native fetal brain. As in Aim 1, immunostaining and RT-PCR will be used to characterize differentiation of DA precursors in 3D cultures. After ex vivo expansion and differentiation of purified NPCs, these hydrogels could potentially be used directly as vehicles for cell transplantation. The innovation of this project lies in the combination of pre-selection for DA precursors prior to ex vivo expansion and subsequent differentiation in 3D, HA biomaterials. The PI hypothesizes that large numbers of DA neurons, sufficient for therapeutic benefit, can be generated using this two-step approach.